1 Type 1 diabetes (T1D) is an autoimmune disease afflicting nearly 2 million people in the U.S. The loss of insulin- 2 producing β cells in the pancreas results in an absolute requirement for injected insulin, causing significant risks 3 of mortality and morbidity. T1D is characterized by a latent (asymptomatic) phase, during which autoimmune or 4 inflammatory pancreatic beta cell injury is postulated to lead to a decline in beta cell function/mass and ultimately 5 to T1D. A key goal in T1D is halting the autoimmune cellular attack, by limiting immune-mediated damage. The 6 precise intrapancreatic signaling mechanisms that lead to activation of the immune system and early pancreatic 7 injury remain unclear. Identification of markers closely associated with these key immune events in the 8 pathogenesis of T1D that can be detected in peripheral circulation would allow for detection of pre-clinical 9 disease and tracking of disease trajectory. Extracellular vesicles (EVs) and their contents have emerged as novel 10 mediators of intercellular signaling and functional biomarkers in human metabolic diseases. Data from our 11 collaborative group as part of NIH efforts in EV biology (NIH Common Fund) suggest that circulating cell-specific 12 EVs and their cargo as probes for disease trajectory or cellular health provide greater specificity than traditional 13 circulating RNA or protein biomarkers in whole plasma. Recent studies in T1D suggest that pancreatic beta cells 14 under “stress” produce EVs containing auto-antigens and RNA transcripts that may mediate communication 15 between pancreatic and immune cells by transfer of molecular cargo. Nevertheless, studies characterizing the 16 functional landscape of pancreatic beta-cell-derived EVs in T1D, and their implications as biomarkers of T1D 17 susceptibility in childhood, are lacking. In response to RFA-DK-21-016, we hypothesize that pancreatic islet cell- 18 derived EVs are functional reporters of islet cell biology and contain RNA cargo relevant to regulation of immune 19 responses and beta cell health early in T1D. In Aim 1, we utilize well-established human cellular systems of 20 pancreatic injury (human cell-line and donated human islets, with and without cytokine-mediated injury) 21 alongside methods established by our group to provide proteomic and transcriptional characterization of beta- 22 cell-derived EVs, with isolation of pancreatic beta-cell specific EVs from human circulation in children with and 23 without T1D. In Aim 2, we define the functional role for pancreatic beta-cell specific EVs in innate immune 24 function (macrophages, neutrophils) postulated to serve as early mediators of pancreatic injury via assessments 25 of immunometabolic phenotypes and responses to in vivo administration of human EVs to a diabetes-prone 26 model system. In Aim 3, we will use RNA-seq and bioinformatics to identify pancreatic beta cell-specific 27 transcripts associated with incident T1D from chil...